KR20210127919A - Streptavidin-oligo conjugate composition of pMHC occupancy - Google Patents

Abstract

The present invention provides a method for determining antigen reactivity and TCR avidity in pMHC multimeric species and biological samples encoded with different nucleic acid molecules, and for sequencing the corresponding T cell transcript, T cell proteome, T cell epigenome or TCR locus. Describe the use.

Description

Streptavidin-oligo conjugate composition of pMHC occupancy

Related applications

This application claims the benefit and priority to U.S. Provisional Patent Application No. 62/781,377, filed December 18, 2018, the contents of which are incorporated herein by reference.

Barcoded antibodies and barcoded pMHC multimers have recently been developed to enable high-troughput sequencing of cognate cells and binding proteins/peptides. Barcoded MHC multimers potentially allow for massively multiplexed NGS-based screening of TCR-pMHC binding events with the ability to combine this information with transcriptomic, proteomic and TCR sequence data at the single cell level. make it Recent examples show the feasibility and utility of barcoded pMHC multimers ^1,4,5 . However, one limitation is the inability of the current technique to fine-tune the pMHC monomer loading per streptavidin to measure TCR-pMHC avidity. There is a need for barcoded pMHC multimers capable of assessing TCR-pMHC avidity. There is also a need for cost-effective methods to produce barcoded pMHC multimers that avoid chemical bonding and save on MHC monomer reagents.

Summary of invention

The present application relates to compositions and methods for producing both covalently and non-covalently linked barcoded SA-oligo conjugates (FIG. 1) that can be used as a platform for combining T cellomics assays in parallel with TCR-pMHC avidity assessment.

In one aspect, the present disclosure provides various backbones (eg streptavidin) consisting of at least one biotinylated pMHC molecule non-covalently bound to a streptavidin molecule and at least one nucleic acid molecule covalently or non-covalently bound to the same backbone molecule. Occupy barcoded pMHC multimer species. wherein the nucleic acid may comprise a central barcode region.

In another aspect, the present disclosure provides barcoding of streptavidin into a biotinylated oligo using HPLC purification to obtain a desired streptavidin occupancy.

In another aspect, the present disclosure provides for barcoding streptavidin with at least one oligo per streptavidin using a covalent bond (eg, a thioethyr bond or a bis-arylhydrazone conjugate bond). do.

In one aspect, the present disclosure provides a pMHC multimer barcoded with at least one nucleic acid molecule comprising a central barcode region and a poly-A tail.

In another aspect, the present disclosure provides a pMHC multimer barcoded with at least one nucleic acid molecule comprising a central barcode region and a nucleotide sequence complementary to a TCR constant gene.

In another aspect, the present disclosure provides a pMHC multimer barcoded with at least one nucleic acid molecule comprising a central barcode region and a template switch oligo sequence.

In another aspect, the present disclosure provides methods of making or using the barcoded pMHC multimers disclosed herein.

In one aspect, the present disclosure provides a peptide-major histocompatibility complex (pMHC) barcoded multimer comprising:

at least one tunable pMHC entity, wherein the pMHC entity comprises at least one pMHC molecule linked by a backbone molecule; and at least one nucleic acid molecule per backbone molecule, wherein said nucleic acid molecule comprises a covalently or non-covalently linked binder:

In some embodiments of the pMHC multimer the nucleic acid molecule comprises:

A central stretch of pMHC barcoded nucleotides and a second stretch of nucleotides with complementarity to the target oligo.

In some embodiments, pMHC multimers are used in NGS.

In some embodiments of the pMHC multimer, the backbone molecule is streptavidin.

In some embodiments of the pMHC multimer, the multimer comprises at least 1, at least 2, at least 3 or at least 4 pMHC bodies per backbone molecule.

In some embodiments, pMHC multimers are used to monitor T cell receptor (TCR)-pMHC avidity.

In some embodiments of the pMHC multimer, the streptavidin is covalently linked to at least one nucleic acid molecule to provide at least 4 MHC monomers per streptavidin.

In some embodiments of the pMHC multimer, streptavidin is non-covalently bound to at least one nucleic acid molecule, wherein the nucleic acid molecule is biotinylated, and wherein the at least one biotinylated nucleic acid molecule and streptavidin are complexed in a ratio. wherein the ratio is selected from the group consisting of 1 streptavidin: 1 oligo, 1 streptavidin: 2 oligo, and 1 streptavidin: 3 oligo.

In some embodiments, the pMHC multimer is prepared by an HPLC purification process.

In some embodiments of the pMHC multimer, streptavidin is covalently bound to at least one nucleic acid molecule, wherein the nucleic acid molecule comprises a barcode and at least one biotin binding site, wherein the binding site comprises:

Biotinylated peptides, biotinylated proteins, biotinylated polymers, biotinylated fluorophores, biotinylated cleavable oligos or biotinylated agents.

In some embodiments of the pMHC multimer, the at least one nucleic acid molecule consists of a 5' PCR handle region, a central barcode region, optionally a UMI, or optionally a 3' poly-A tail region of at least 10 contiguous adenines.

In some embodiments of the pMHC multimer, the at least one nucleic acid 3' terminal tail consists of any sequence complementary to the target oligo sequence.

In some embodiments of the pMHC multimer, the at least one nucleic acid molecule is about 10-200 nucleotides or more in length.

In another aspect, the present disclosure provides a composition comprising a plurality of subsets of any of the aforementioned pMHC multimers, wherein each subset of the pMHC multimers binds a different peptide and has a corresponding barcode region sequence.

In another aspect, the present disclosure provides a method of linking a particular MHC molecule with a corresponding T cell transcript, comprising:

a) forming a test sample comprising a plurality of any of the foregoing pMHC multimeric molecules, T cells and particles linked to a binding target oligo comprising a 5' PCR handle, a central cell barcode, a UMI, and a bait sequence; step;

b) forming droplets from the test sample such that each droplet contains only one particle and one T cell bound to one or more pMHC multimeric molecules;

c) generating a T cell cDNA library and a pMHC barcode library from each drop; and

d) sequencing both the T cell mRNA library and the MCH barcode library to link specific MHC molecules with the corresponding T cell transcript.

In some embodiments of the method, the bait sequence is 3' poly-(dT).

In another aspect, the present disclosure provides a multimeric pMHC comprising:

one or more pMHC molecules linked by backbone molecules; and at least one nucleic acid molecule linked to said backbone, wherein said nucleic acid molecule is a nucleotide sequence complementary to a central stretch (barcode region) of a nucleic acid designed to be amplified and a TCR constant gene.

In some embodiments, the multimeric pMHC comprises a type 1 nucleic acid molecule linked to a backbone; wherein the nucleic acid molecule of type 1 comprises a central barcode region and a nucleotide sequence complementary to a TCRα or TCRβ constant gene.

In some embodiments, the multimeric pMHC comprises nucleic acid molecules of type 1 and type 2 linked to a backbone; here

The type 1 nucleic acid molecule comprises a central barcode region and a nucleotide sequence complementary to the TCRα constant gene,

The type 2 nucleic acid molecule comprises a central barcode region and a nucleotide sequence complementary to the TCRβ constant gene;

The barcode regions of the two types of nucleic acid molecules have the same sequence;

Optionally, the UMI sequences for each of the two types of nucleic acid molecules are random and thus differ from each other even if they are located in the same region of each nucleic acid.

In some embodiments of the multimeric pMHC, the nucleic acid molecule comprises a 3' nucleotide sequence complementary to a 5' PCR handle, a central barcode region, UMI and TCR constant genes.

In some embodiments of the multimeric pMHC, the nucleic acid molecule comprises a nucleotide sequence complementary to the 5' end of the TCR constant gene.

In some embodiments of the multimeric pMHC, the TCR constant gene is a TCRα constant gene, a TCRβ constant 1 gene, or a TCRβ constant 2 gene.

In some embodiments of the multimeric pMHC, the 5'-terminal and/or 3'-terminal nucleic acid molecule is linked to a backbone molecule.

In some embodiments of the multimeric pMHC, the nucleic acid molecule further comprises a unique molecular identifier (UMI) adjacent to the barcode region.

In some embodiments of the multimeric pMHC, the at least one nucleic acid molecule is about 10-200 nucleotides or more in length.

In another aspect, the present disclosure provides a composition comprising:

a plurality of subsets of any of the aforementioned multimeric pMHCs, wherein each subset of the multimeric MHCs binds a different peptide and has a corresponding barcode region sequence.

In another aspect, the present disclosure provides a method of linking a specific MHC molecule with a corresponding TCRα and/or TCRβ sequence, comprising:

a) providing one or more of any of the aforementioned multimeric pMHCs;

b) contacting said multimeric pMHC molecule with a T cell;

c) isolating the T cells bound to the multimeric MHC molecule from the cells not bound to the multimeric MHC molecule;

d) lysing the isolated T cells;

e) generating a DNA library in which each DNA molecule comprises a sequence of TCRα and/or TCRβ genes and a pMHC barcode; and

f) sequencing the DNA library to link specific pMHC molecules to the corresponding TCRα and/or TCRβ sequences.

In some embodiments of the method described above, step c) is accomplished by FACS sorting or magnetic bead-based separation.

In some embodiments of the aforementioned methods, the multimeric pMHC molecule is directly or indirectly fluorescently labeled.

In some embodiments of the aforementioned methods, the T cells bound to the barcoded pMHC molecules are bulk sorted in a single collection tube.

In some embodiments of the aforementioned methods, cognate T cells associated with the barcoded pMHC molecule are sorted into individual plate wells as single cells.

In another aspect, the present disclosure provides a multimeric pMHC comprising:

one or more pMHC molecules linked by backbone molecules; and at least one nucleic acid molecule linked to the backbone, wherein the nucleic acid molecule comprises a central stretch of a nucleic acid (barcode region) designed to be amplified and a template switch oligo sequence.

In some embodiments of the multimeric pMHC, the nucleic acid molecule comprises a 5' PCR handle, a central barcode region, a UMI and a 3' template switch oligo sequence.

In some embodiments of the multimeric pMHC, the template switch oligo sequence comprises a 3' stretch of three riboguanosines.

In some embodiments of the multimeric pMHC, at least one nucleic acid molecule is about 10-200 nucleotides or more in length.

In another aspect, the present disclosure provides a composition comprising:

a plurality of subsets of any of the aforementioned multimeric pMHCs, wherein each subset of multimeric pMHCs binds a different peptide and has a corresponding barcode region sequence.

In another aspect, the present disclosure provides a method of linking a particular pMHC molecule to a corresponding TCRα and/or TCRβ complementary sequence, comprising:

a) a test comprising beads bound to a plurality of any of the aforementioned multimeric pMHC molecules, T cells, and oligos comprising a 3' nucleotide sequence complementary to a 5' PCR handle, a central cell barcode, UMI and TCR constant genes forming a sample;

b) forming droplets from the test sample such that each droplet contains only one bead and one T cell bound to one or more MHC multimeric molecules;

c) generating a DNA library, wherein each DNA molecule comprises a sequence of TCRα and/or TCRβ genes, and a pMHC barcode; and

d) sequencing the DNA library to link specific pMHC molecules to the corresponding TCRα and/or TCRβ sequences.

In some embodiments of the method, the beads are selected from hydrogel beads, hard beads and soluble beads.

In some embodiments of the method, the bead is linked to two oligos, wherein the first oligo comprises a 3' nucleotide sequence complementary to a 5' PCR handle, a central cell barcode, UMI and TCRα constant genes; the second oligo contains a 3' nucleotide sequence complementary to the 5' PCR handle, central cell barcode, UMI and TCRβ constant genes; The central cell barcodes for the two oligos have identical sequences.

In some embodiments of the method, step c) of generating the DNA library comprises reverse transcription of TCR mRNA using MMLV reverse transcriptase.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the PCR handle enables library preparation of barcode sequences.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the PCR handle may have an i7 adapter sequence.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the barcode region comprises at least 4 nucleotides.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the pMHC molecule is attached to the backbone via a streptavidin-biotin bond, via an MHC heavy chain, or via an MHC light chain (β2M). Connected.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the MHC molecule is linked to the backbone via a streptavidin-biotin bond.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the multimeric pMHC comprises at least one MHC molecule.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the at least one nucleic acid molecule further comprises a chemical modification.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the 5' or 3' terminus of at least one nucleic acid molecule is attached to an amino group via a spacer.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the spacer can be a 6-carbon spacer or a 12-carbon spacer.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the at least one nucleic acid molecule comprises phosphorothioated nucleotides at the 5' and/or 3' ends.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the linkage between the at least one nucleic acid molecule and the backbone molecule allows for inducible dissociation of the nucleic acid molecule.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the at least one nucleic acid molecule is linked to the backbone molecule via a covalent or non-covalent bond.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the covalent bond is a thioether.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the at least one nucleic acid molecule is linked to the backbone molecule via an inducibly cleavable bond.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the inducibly cleavable bond is photocleavable or comprises a disulfide bond.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the MHC molecule is an MHC class I and/or MHC class II monomer.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the MHC molecule is complexed with a peptide.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the MHC molecule is biotinylated.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the backbone further comprises one or more labels selected from the group consisting of a fluorescent label, a His-tag, and a metal-ion tag.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the backbone is directly associated with a fluorescent label.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the fluorescent label is a fluorophore-tagged oligo.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the fluorophore-tagged oligo is complementary to a nucleic acid molecule linked to the backbone.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the backbone is labeled with a fluorophore labeled anti-streptavidin antibody.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the fluorophore is a fluorescent protein, a fluorescent dye, or a quantum dot.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the at least one nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of DNA, RNA, artificial nucleotides, PNA and LNA.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the multimeric pMHC binds to a cognate T cell.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the multimeric pMHCs are compatible with flow cytometry applications.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the flow cytometry application is single cell or bulk cell fluorescence-activated cell sorting (FACS).

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the multimeric pMHCs are compatible with NGS-based applications.

In some embodiments of any of the aforementioned multimeric pMHCs and methods, the NGS-based application is droplet-based single cell sequencing.

In another aspect, the present disclosure provides a method of detecting antigen-reactive cells in a sample comprising:

a) providing one or more of any of the foregoing multimeric pMHCs;

b) contacting said multimeric pMHC molecule to said sample; and

c) detecting the binding of the multimeric pMHC molecule to the antigen-reactive cell, thereby detecting a cell responsive to the antigen present in the MHC molecule, wherein the binding of the nucleic acid molecule is linked to one or more MHC molecules via a backbone molecule. It is detected by amplifying the barcode region.

In some embodiments of the method, the sample is selected from the group consisting of a blood sample, a peripheral blood sample, a blood-derived sample, a tissue sample, bodily fluid, spinal fluid, and saliva.

In some embodiments of the method, the sample is obtained from a mammal.

In some embodiments of the method, the method further comprises selecting the cells by a method selected from the group consisting of flow cytometry, FACS, magnetic-bead based selection, size-exclusion, gradient centrifugation, column attachment, and gel filtration.

In some embodiments of the method, the amplification is PCR.

In some embodiments of the method, detecting the barcode region of the nucleic acid molecule comprises sequencing the barcode region or detecting the barcode region by qPCR.

1 shows the general structure of a modified oligo for binding to streptavidin to prepare barcoded pMHC multimers. Purple bars represent oligos and green stars represent 5' modifications. Below is the oligo sequence used for covalent bonding. The 5' end of the oligo is an amino group with a 12-carbon spacer, a 6-carbon spacer may also be used. Alternative oligo modifications may be used in place of amino groups including, but not limited to, thiol groups or biotin groups. The 5' biotin group can be used for non-covalent binding to streptavidin. Such oligo modifications may also be included at the 3' end of the oligo. The PCR handle enables library preparation of barcode sequences, and in this particular case, the PCR handle is an i7 adapter sequence. The tetrameric barcode sequence is the sequence that follows the PCR handle and corresponds to a given pMHC complex. The pMHC barcode in this sequence consists of 6 nucleotides allowing up to 4096 tetrameric barcode possibilities. Longer pMHC barcode sequences can be used for increased throughput. At the 3' end of the oligo is a poly-A tail capable of binding to the target sequence, in this case poly(dT)VN. For this oligo, a longer adenine stretch can be used, but 25 adenines are used. The B nucleotide (G, C or T) at the 5' end of the poly-A tail allows the V nucleotide (G, C or A) to bind second to the last 3' nucleotide of the poly(dT)VN . If the N nucleodides (all bases) of the bait sequence are C, then complementarily binds to the depicted oligo sequence. An optional phosphorothioated DNA base at the 3' end of the oligo provides protection from exonuclease activity. The deformed base can be placed in any position.
Figure 2 shows the binding of barcoding oligos to streptavidin with a disulfide bridge interposed therebetween. In the example presented, binding can be performed using a Solulink Protein-Oligonucleotide Conjugation Kit (TriLink Biotech). Streptavidin (in this case Prozyme) is transformed into succinimidyl-6-hydrazino-nicotinamide (S-HyNic). The 5'-amino-modified oligo is modified with a succinimidyl-4-formylbenzamide homologue (S-SS-4FB). The modified streptavidin and the modified oligo are combined to produce barcoded streptamidine. An alternative methodology involves linking the oligo to streptavidin with an intermediate photocleavable linker (not shown).
Figure 3 shows that DNA barcoding oligos dissociate from streptavidin under denaturing conditions. A 66-mer oligo bound to streptavidin with an intermediate disulfide bridge is run on a SYBR-safe agarose gel (lane 1a). Excess oligos are shown below the 100 bp ladder band. In lane 2a, the same amount of barcoded streptavidin as in lane 1a was incubated with beta-mercaptoethanol for 20 minutes before loading on the gel. The band representing barcoded streptavidin is severely reduced. The same gel was stained with Coomassie Blue to visualize the protein. In lane 1b, most of the protein in lane 2b migrated to the top of the gel, while bacodinated streptavidin was evident. Without the negative charge of the oligo, bare streptavidin moves much more slowly.
4 shows that barcoded streptavidin retains the ability to bind biotin. Equal amounts of barcoded streptavidin were incubated with biotin magnetic beads (lane 1a) or streptavidin magnetic beads (lane 2a). Equal amounts of biotin magnetic beads or streptavidin magnetic beads were used in this experiment. Samples were applied to a magnetic separator to separate bound versus unbound barcoded streptavidin. All unbound fractions in both reactions were harvested and run on SYBR-Safe agarose gel. The same gel was stained with Coomassie Blue to visualize the protein (lanes 1b and 2b). Consumption of barcoded streptavidin (lanes 1a and 1b) indicates biotin binding. Both biotin and streptavidin magnetic beads were purchased from RayBiotech Inc.
5 shows barcoded pMHC multimeric variants. The left-most barcoded pMHC multimer consists of a covalently derived streptavidin-oligo conjugate, after which the biotinylated pMHC monomer is used to tetramerize according to established techniques. Such covalent bonds can be derived from many bonding chemistries, including but not limited to thioether bond formation.
All other conjugates utilize a non-covalent linkage between the biotinylated oligo and streptavidin. Second from the left, the barcoded pMHC multimer consists of purified streptavidin-oligo conjugates in a ratio of 1:1 (1b) followed by pMHC trimerization. Third from left, barcoded pMHC multimer consists of purified streptavidin-oligo conjugate in a 1:2 (2b) ratio, followed by pMHC dimerization. On the far right, the barcoded pMHC multimer consists of a purified streptavidin-oligo conjugate in a 1:3 ratio, which is then combined with pMHC monomer to give a 1:1 molar ratio of streptavidin:monomer. Different colored oligos represent different barcode sequences for each multimeric species. The lower triangle represents the predicted TCR-pMHC binding capacity of each multimerized binder, 4 monomers > 3 monomers > 2 monomers > 1 monomer.
Figure 6 includes four panels, Figures 6a-6d showing the methodology of barcoded pMHC multimer detection. In Figure 6a, a fluorophore-containing streptavidin (eg PE-streptavidin) is used as a starting point for further binding to the oligo. In Figure 6b, a fluorophore-tagged oligo complementary to the bound oligo is annealed to the pMHC barcoded oligo. The length of this annealing oligo may vary. In Figure 6c, a fluorophore-labeled anti-streptavidin antibody is used to detect barcoded pMHC multimers. Species of fluorophores used include, but are not limited to, fluorescent dyes and quantum dots. Barcoded pMHC multimeric species to which oligos are non-covalently linked can also be detected using this method. Figure 6d shows the possibility of individually labeling different pMHC multimeric species (including the same peptide) with different fluorescent dyes containing anti-streptavidin antibodies. One of the three fluorescent labeling techniques of FIGS. 6A-6C can be applied. In this way, other TCRs can be screened for binding to this particular pMHC protein complex using flow cytometry. In this particular situation, the four monomeric pMHC multimers (tetramers) do not require covalently linked oligos, and the pMHC multimeric species that utilize non-covalently linked oligos, provided that all that is needed is that the oligos block the biotin binding site. , does not require different oligo barcode sequences. Other biotinylated molecules could theoretically be used to block the biotin binding site as described in FIG. 14 .
7 shows an example of application using barcoded pMHC multimers for monitoring immuno-epitope avidity. Tumor-derived mutant gene-encoding proteins are tiled peptides for TCR sequencing studies. In this description, two overlapping peptide amino acid sequences (indicated by numbers) of the same mutant protein are individually biotinylated MHC (same MHC allele) followed by barcoded streptavidin conjugates as shown. form a complex Oligo sequences for all types of binders, regardless of pMHC, contain a unique barcode sequence that distinguishes them (indicated by colored lines attached to streptavidin). The barcoded pMHC multimer is mixed with the sample. TCR alpha and beta sequences are obtained via single-cell RNA sequencing and are paired with cognate pMHC multimeric species. For example, one study using such a conjugate can determine whether a green peptide-MHC complex or a red peptide-MHC complex has higher avidity to the cross-linked TCR sequence.
8 shows another application example of barcoded pMHC multimer immune-epitope avidity monitoring. In this hypothetical example, the vial contains a defined mixture of barcoded pMHC multimeric species consisting of a streptavidin: oligo ratio of 1:1, 1:2 or 1:3, which is a streptavidin:pMHC monomer. The molar ratio may be defined as 1:3, 1:2 and 1:1 respectively. Oligos for the binder species contain unique barcode sequences (sequences of different colors) that can distinguish them. Since TCR-pMHC avidity is known to decrease within the tumor microenvironment, these barcoded pMHC multimeric conjugate species can be used in therapeutic procedures, eg. It is expected that this may explain the increased or decreased TCR binding capacity during Immune Checkpoint Blockade (ICB) treatment. In the given example, after ICB treatment, T cells have increased avidity with cognate pMHC complexes as evidenced by higher read counts of multimers comprising 1 and 2 pMHC monomers.
Figure 9 shows the preparation of non-covalently linked barcoded pMHC multimers for detection via NGS. Biotinylated oligos (containing pMHC multimer-specific barcodes) are mixed with streptavidin (or streptavidin-fluorophore conjugate) in a ratio of 0.5-1.0 oligo to 1 streptavidin. The 1:1 SA: oligo conjugate is precisely purified via HPLC to remove unreacted biotinylated streptavidin, unreacted biotinylated oligo and other SA: oligo ratio conjugates. The conjugate is then multimerized with the biotinylated pMHC monomer. The final product is a barcoded pMHC trimer in which each streptavidin has one oligo and three pMHC monomers. The same concept is used when preparing conjugates composed of 1:2 and 1:3 streptavidin:oligo ratios.
Figure 10 shows that barcoded pMHC of different monomer ratios can similarly detect T cells when used at sufficiently high concentrations and when detecting TCRs of moderate avidity. Streptavidin-oligo conjugates were prepared by linking biotinylated oligos to streptavidin in an SA:oligo ratio of 1:1 or 1:2. Streptavidin alone was used as a control, which has 4 pMHC monomers per streptavidin (tetramer) as described in the figure legend below. 1: 1 SA: oligo conjugate contains 3 monomers while 1: 2 SA: oligo conjugate contains 2 monomers. For the purposes of this experiment, the oligos for all barcode binders consisted of the same 69mer nucleotide sequence. The left three columns show ^{multimeric staining with HLA-A *} 11: 01 MHC I complexed with CMV pp65 peptide, followed by streptavidin, one oligo and streptavidin or two oligos and streptavidin. They multimerized together. ^{The middle three columns show multimeric staining with HLA-A *} 11: 01 MHC I complexed with EBV 399-408 peptide. ^{The right three columns show multimer staining using HLA-A *} 11: 01 MHC I complexed with EBV 416-424 peptide. The amount of streptavidin used for each stain is indicated on the far left of the line. The percentage of multimer positive staining is indicated in each box. Previously, peptide expanded cells were used to stain with anti-CD3, anti-CD8, live/dead dyes and non-fluorophore barcoded pMHC multimers (or tetramers). Secondary staining consisted of anti-streptavidin-PE to detect pMHC multimers. The flow cytometry gate legend is shown at the top of the figure.
Figure 11 shows that barcoded pMHC multimers of different oligo and monomer ratios can discriminate pMHC-TCR avidity. Streptavidin-oligo conjugates were prepared by covalently linking oligos to streptavidin via covalent bonds (thioether bonds) or by linking biotinylated oligos to streptavidin at various SA:oligo ratios. For the purposes of this experiment, the oligos for all conjugates consisted of the same 46mer nucleotide sequence. A covalent bond represents one streptavidin molecule bound to one oligo with all four biotin binding sites available. The 1b conjugate represents one streptavidin linked to one biotinylated oligo with three biotin binding sites remaining. The 2b conjugate shows one streptavidin bound to two biotinylated oligos with two biotin binding sites remaining. The 3b conjugate represents one streptavidin linked to three biotinylated oligos with one biotin binding site remaining. H-2Kb biotinylated monomer (MBLI) was complexed with either the high affinity SIINFEKL peptide or the low affinity SIIVFEKL peptide. In this case, affinity refers to the strength of the interaction between the pMHC complex and the OT-I TCR of the transformation, not the interaction between the MHC and the peptide. Avidity in this case refers to the bound affinity when several pMHCs are involved in the evaluation of TCR binding.
The pMHC molecule forms a complex with a streptavidin-oligo complex to produce a barcoded pMHC multimer. OT-I splenocytes were stained with various barcoded H-2Kb barcoded pMHC multimer species with varying amounts of SA (0.25 ug first row, 0.1 ug second row, 0.025 ug third row). The covalent bond is pMHC tetramerized using a molar ratio of 1 SA to 4 monomers. The 1b conjugate is Pmhc trimerized using a molar ratio of 1 SA to 3 monomers. The 2b conjugate is pMHC dimerized using a molar ratio of 1 SA to 2 monomers. The 3b conjugate is pMHC bound using a molar ratio of 1 SA to 1 monomer. Cells were stained simultaneously with anti-CD3, anti-CD8, live/dead dyes and each non-fluorophore barcoded pMHC multimeric species. Secondary staining consisted of anti-streptavidin-PE to detect pMHC multimers. The flow cytometry gate legend is shown at the top of the figure. The percentage of pMHC multimer positive staining is indicated in each box as low, medium or high intensity.
12 shows the results of a hypothetical experiment using barcoded pMHC multimers of different pMHC occupancy to discriminate pMHC avidity to TCR. SIINFEKL and SIIVFEKL peptide-derived barcoded pMHC multimers are depicted mixed with OT-I splenocytes (left) in a virtual experimental environment. The type ratio of each multimer can be adjusted according to the experimental goal. Alternatively, aliquots of cells can be individually incubated with SIINFEKL or SIIVFEKL barcoded pMHC multimers. Each multimeric species has a unique barcode sequence to distinguish it from other multimeric species. FACS sorting can be used prior to droplet-based single cell sequencing or other single cell sequencing platforms (middle). On the right is the predicted barcode reading frequency. In this case, high affinity/avidity SIINFEKL-based pMHC barcode reads predominate in all monomeric species, whereas low affinity/avidity SIIVFEKL-based pMHC barcode reads can only appear in 4 monomeric species. For this particular situation, the use of only four monomer-based pMHC multimeric species for each peptide (SIINFEKL versus SIIVFEKL) would be sufficient to determine a pMHC complex with higher avidity for this particular TCR. However, where the avidity differences are more subtle and different TCRs are involved, the use of pMHC barcoded species could potentially reveal differences in avidity.
13 shows that barcoded pMHC multimers can be generated with directly bound fluorophores. Streptavidin-oligo conjugates were prepared by covalently linking oligo-streptavidin via thioether linkages (Cov) or linking biotinylated oligos to streptavidin in a ratio of 1 SA:1 oligo (1b). . In addition, a fluorophore (Alexa Fluor 647) comprising an SA-oligo conjugate was prepared. In this case, AF647 was first bound directly to streptavidin, then the oligo was directly bound to streptavidin. For the purposes of this experiment, the conjugates consisted of identical 69mer nucleotide sequences. A covalent bond represents one streptavidin molecule bound to one oligo with all four biotin binding sites available. The 1b conjugate shows one streptavidin bound to one biotinylated oligo with three biotin binding sites remaining. The combination is in the indicated proportion HLA-A ^* 02: 01 monomer biotinylated CMV pp65 peptide NLVPMVATV or negative control HLA-A ^* 02, comprising: a large amount has been embodied to 01 monomers (MBLI). ^{Human HLA-A*} 02:01 PBMCs previously expanded with CMV pp65 peptide (NLVPMVATV) were used to stain with binders. Cells were stained with anti-CD3, anti-CD8, live/dead dyes and barcoded pMHC multimers. Middle column samples were stained with MBLI commercial CMV pp65 HLA-A ^* 02: 01 PE tetramer as control. Samples containing conjugates without fluorophores were stained secondarily with anti-SA-PE. The flow cytometry gate legend is shown at the top of the figure. Percent pMHC multimer positive staining is indicated in each box.
14 shows an alternative method for generating barcoded pMHC multimeric species. All conjugate species shown carry one oligo per streptavidin via a covalent bond (eg a thioether bond). In a real-world experimental setting, each conjugate species (4, 3, 2 or 1 monomer) has a unique barcode sequence marked with a different color. Biotinylated peptides, biotinylated proteins, biotinylated lipids, biotinylated fluorophores, biotinylated polymers or cleavable biotinylated oligos (all possibilities are usually marked in purple) are optimized Incubated with SA-oligo conjugates at the specified ratio and HPLC-purified to the desired biotin occupancy. This purified conjugate is then multimerized with biotinylated pMHC monomer to completely occupy the biotin binding site per streptavidin. The lower triangle represents the predicted TCR-pMHC binding capacity of each multimerized binder, 4 monomers > 3 monomers > 2 monomers > 1 monomer.
15 shows bulk pMHC multimer barcode sequencing for immuno-epitope dominance analysis. In this embodiment, barcoded pMHC multimers (stars with different colored lines) are quantified from cells with or without magnetic beads or enrichment such as FACS. In the example shown, five different gp100 peptides were made with separate barcoded pMHC multimers, with each gp100 peptide/MHC complex associated with a unique multimeric barcode (marked in a different color). Barcoded pMHC multimers may use covalently or non-covalently linked oligos. Oligos are processed for library preparation and sequencing.
For applications that require small barcoded oligo libraries (eg 8 or less), pMHC multimeric barcode quantification can be confirmed without the use of sequencing. Fluorescently labeled oligos complementary to the pMHC multimeric region can be pre-annealed prior to incubation with cells. Each barcoded pMHC multimer type will anneal to a separate fluorophore containing oligos prior to incubation with cells and subsequent flow cytometry analysis. For example, pMHC multimers comprising gp100 #1 peptide and related oligo sequences are pre-annealed to the complementary AlexaFluor488-oligo, pMHC multimers comprising gp100 #2 peptide and related oligo sequences are complementary to AlexaFluor532-oligos, etc. pre-annealed.
16 shows the use of barcoded pMHC multimers with an NGS-based single cell sequencing platform. The barcoded pMHC multimer (covalently linked oligo in this example) bound to the target cell is captured in a single droplet. Most individual droplets contain one T-cell/barcoded pMHC complex with particles containing the targeting oligo. The barcode oligo in this example comprises poly-A. Both oligo and T cell mRNA are reverse-transcribed for further library preparation. Optionally, a cleavable bond (UV or disulfide) between the oligo and streptavidin can be introduced during the association of the oligo and streptavidin. In particular, disulfide bonds will dissociate in the droplet due to the reducing environment of the lysis buffer.
17 includes eight panels. 17A-17H show TCR loci targeting barcoded pMHC multimers to specifically sequence TCR and pMHC complex reads. An alternative to oligo design in bulk sequencing and droplet-based NGS platforms is to have oligos targeting the desired endogenous mRNA transcript while maintaining the identity of the pMHC complex. Various designs have been shown for barcoded pMHC multimers to specifically examine either TCR clonality or single cell TCR repertoires with their corresponding pMHC identity. This design allows for the sequencing of TCR transcripts along with the pMHC barcode in one read. The PCR handle contains the region to which the PCR primers bind for amplification. Barcode (BC) indicates the nucleotide sequence that identifies the pMHC complex. The TCR complementary sequence (TCR compl) is complementary to the TCR constant gene. The TCR complementary sequence serves as a primer during reverse transcription.
In Figure 17a, the barcoded oligo is covalently linked to streptavidin to produce a barcoded tetramer. This barcoded tetramer contains a 3' sequence complementary to the TCRβ constant region gene. Since there is significant sequence similarity between the two constant genes at the 5' end (closest to each J gene), the TCRβ constant gene oligo sequence is complementary to both the TCRβ constant 1 gene and the TCRβ constant 2 gene. The purified barcoded streptavidin conjugate is then tetramerized with pMHC. A unique molecular identifier (UMI) can also be placed near the tetrameric barcode for downstream PCR deduplication, allowing for more accurate pMHC binding quantification. TCRβ targeting oligos alone can be used to study TCR clonality. Alternatively, the TCRα constant gene targeting the oligo can be used alone (not shown).
In FIG. 17B , the streptavidin-oligo conjugate purified from A is further modified to include a covalently linked oligo targeting the TCRα constant gene. This construct is suitable for a single cell sequencing platform to obtain the complete TCRα/β sequence (same as Figure 17d-h). This double oligo streptavidin conjugate is then tetramerized.
In Figure 17c, TCRβ targeting biotinylated oligos is non-covalently attached to streptavidin, purified and then trimerized with biotinylated pMHC monomers.
In Figure 17d, the purified TCRβ conjugate from c is non-covalently attached to streptavidin, purified and then dimerized with biotinylated pMHC monomer.
In Figure 17e, a single oligo containing both TCRβ and TCRα target sequences and biotinylated at both ends is used. The desired conjugate is purified and dimerized with biotinylated pMHC monomer. Alternative oligo designs for this include a cleavable (UV or disulfide) bond between the two halves.
In Figure 17f, a single oligo biotinylated at both ends containing both TCRβ and TCRα target sequences is used. The sized streptavidin-oligo conjugate was purified and 2x trimerized with biotinylated pMHC monomer to give a total of 6 pMHC monomers for the oligosaccharide. 17e, this design can be further reinforced to include a cleavable (UV or disulfide) bond between the two halves.
In Figure 17G, a single oligo comprising both TCRb and TCRa targeting sequences is covalently attached to streptavidin. The purified conjugate is tetramerized with biotinylated pMHC monomer. 17E and 17F , this binder may include a cleavable bond.
In FIG. 17H , a single biotinylated oligo comprising both TCRb and TCRa targeting sequences can be non-covalently attached to streptavidin. The purified conjugate is trimerized with biotinylated pMHC. As shown in Fig. 17e-g, this binder may contain a cleavable bond.
18 shows TCR locus target barcoded pMHC multimers used for FACS sorted single cell TCR clonal or paired TCRa/b sequencing. Although only a few designs are shown for illustrative purposes, any barcoded pMHC multimer design in the preceding figures can be used.
A barcoded tetramer containing a TCRβ constant gene targeting oligo is shown on the left. Alternatively, TCRα constant gene targeting oligos can be used or both can be used with the design described in the previous figures. This complementary TCR α/β oligo sequence will serve as a reverse transcription primer for the endogenous TCRα/β mRNA transcript. To utilize TCR targeting barcoded pMHC multimers in cell sorting applications, fluorescence can be incorporated as described in FIG. 6 . T cells are first sorted into single cells and reverse transcribed in-well, then processed in bulk in single tubes for clonal studies. T cell #1 is bound by a barcoded pMHC multimer comprising barcode sequence #1 while T cell #2 is bound by a barcoded pMHC multimer comprising barcode sequence #2. On the right, the barcoded pMHC multimer contains both TCR α and β targeting sequences as described in the previous figure. T cells are sorted into single cells and treated in individual plate wells, in this case 96 well plates.
Clonal studies allow researchers to understand the breadth of TCR usage for a given pMHC complex. A disadvantage to bulk sequencing if both TCRα and TCRβ barcoded oligos are used is that the TCRα and TCRβ sequences cannot pair. To overcome this problem, T cells can be treated as shown at right to allow pairing of TCRα and TCRβ sequences.
The library preparation strategy is described below, and is expanded from previous figures:
i) Single sorted T cells are reverse transcribed and then processed in bulk. RNase treatment is followed by bridge adapter ligation. Bridge adapters are common to all reverse transcribed genes. Subsequent PCR amplification produces the final library preparation.
ii) Single sorted cells are reverse transcribed by SMARTScribe RT (Clontech). SMARTer first-strand synthesis and RT template conversion generate 5'-RACE Ready cDNA. Subsequent PCR amplification of large amounts produces the final library preparation.
iii) Single cell sorted T cells are lysed and intra-well RT, intra-well RNase treatment followed by intra-well bridge adapter ligation. The bridge adapter upstream sequence is common to all wells, but all wells receive a bridge adapter with a unique cellular barcode at the site closest to the TCR sequence. The main advantage of this methodology is that TCRα and TCRβ sequences occurring in the same cell can be paired, allowing the complete TCR sequence of an individual T cell to be identified. Ligated transcripts are pooled for subsequent PCR amplification using common primers to create a final library preparation.
iv) Single sorted T cells are lysed and subjected to in-well SMARTScribe RT (Clontech). SMARTer first-strand synthesis and RT template conversion generate 5'-RACE-prepared cDNA. To individually barcode each well, intra-well PCR amplification is performed using a forward primer complementary to the SMARTerIIA oligo with a unique cellular barcode sequence upstream of the SMARTerIIA binding sequence. This will distinguish transcripts from individual wells. A consensus priming site (ie, i5 sequence) at the 5' end of the forward primer allows for sample pooling. Pooled PCR amplification using consensus primers produces the final library preparation. As with iii, the main advantage of this methodology is that TCRα and TCRβ sequences originating from the same cell can be paired.
19 shows TCR target barcoded pMHC multimer library preparation for clonal studies using bridge adapters. As illustrated in Figure 18(i), barcoded pMHC multimers bound by cognate T cells are first sorted into single cells, reverse transcribed and then mass processed. For display, both TCRα and TCRβ derived transcripts are shown, but only one should be used for clonal studies. The use of the fluorophore tracking strategy described in Figure 6 is required for FACS sorting. Intra-well RT followed by RNase treatment followed by bridge adapter ligation. The bridge adapter for bulk sequencing (i5 sequence) is identical for all cells and contains a 5' phosphate in the lower oligo of the bridge adapter required for ligation to the 3' end of the newly reverse-transcribed TCR transcript. The bridge adapter also contains 6 random nucleotides at the 3' end of the unlinked strand to improve the stability of the adapter linkage. After ligation, PCR amplification using common primers produces the final library preparation.
20 shows TCR target barcoded pMHC multimer library preparation for bulk sequencing using SMARTScribe enzyme (Clontech). As illustrated in Figure 18(ii), barcoded pMHC multimers bound by cognate T cells are first sorted into single cells, reverse transcribed and then mass processed. For display, both TCRα and TCRβ derived transcripts are shown, but only one should be used for clonal studies. The use of the fluorophore tracking strategy described in Figure 6 is required for FACS sorting. In-well RT with SMARTScribe enzyme adds SMARTerIIA oligo sequence via template conversion to the 3' end of the newly reverse-transcribed transcript. After template conversion, bulk PCR amplification using common primers produces the final library preparation.
21 shows TCR target barcoded pMHC multimer library preparation for single cell pair TCRα/β sequencing using bridge adapters. As illustrated in Figure 18(iii), barcoded pMHC multimers bound by cognate T cells sort single cells into individual wells. The use of the fluorophore tracking strategy described in Figure 6 is required for FACS. Sorted cells are lysed and subjected to intra-well RT, intra-well RNase treatment and intra-well bridge adapter ligation (only single TCR transcript shown in ligation step for simplicity). A bridge adapter (eg, i5 sequence) for each well contains a unique cellular barcode that allows pairing of TCRα/β sequences. In this figure, 00001 represents the cell barcode of a specific well. A 5' phosphate on the ligating strand of the bridging adapter enables ligation to the 3' end of the newly reverse-transcribed TCR transcript. The bridge adapter also contains 6 random nucleotides at the 3' end of the unlinked strand to improve the stability of the adapter bond. After ligation, samples are pooled for PCR amplification using common primers to create a final library preparation.
22 shows TCR target barcoded pMHC multimer library preparation for single cell sequencing using SMARTScrib enzyme. As illustrated in Figure 18(iv), barcoded pMHC multimers bound by cognate T cells sort single cells into individual wells. The use of the fluorophore tracking strategy described in Figure 6 is required for FACS. Sorted cells are lysed and subjected to intra-well RT using SMARTScribe enzyme (Clontech), which via template switching adds the SMARTerIIA oligo sequence to the 3' end of the newly reverse-transcribed transcript. Afterwards, intra-well RCR amplification (only single transcripts shown for simplicity) was performed using forward primers containing both the binding sequence as well as the cell barcode sequence (unique to all wells) and a consensus i5 sequence at the 5' end. do. In this figure, the cell barcode is indicated by 00001. The use of forward primers containing unique cellular barcodes enables pairing of TCRα/β sequences. For further PCR amplification, samples are pooled to create a final library preparation.
23 includes two panels. 23A-23B show barcoded pMHC multimers containing switch oligos for droplet-based single cell TCRα/β single cell sequencing. pMHC multimers with covalently linked oligos comprising a switch sequence in FIG. 23A . pMHC multimer with non-covalently linked oligos comprising a switch sequence in FIG. 23B . Additional designs for this type of pMHC multimer include, but are not limited to, those described in FIGS. 5 and 10 .
24 shows droplet-based sequencing beads (including but not limited to hydrogel beads, hard beads or soluble beads) bound to oligos composed of PCR handles, cell barcodes (BC) and TCR constant gene complementary sequences. shows the use of For simplicity, only one drop is shown. Each bead may include TCRα and/or TCRβ targeting oligo sequences. If both TCRα and TCRβ type oligos are used, both will contain the same cell barcode for a given bead. TCRα and/or TCRβ oligos can be dissociated by one of several means including, but not limited to, photocleavage bonds or disulfide bonds. In addition, each oligo may include a UMI for PCR deduplication.
A single drop containing a single bead and a single cell (in this case barcoded tetrameric-positive T cells are indicated) is a lysis reagent that releases both gel bead oligos, T-cell mRNA and tetrameric-positive T-cell-associated barcoded oligos. will include pMHC tetrameric binding oligos can also be made to dissociate by cleavable bonds. Reverse transcription adds the TCR V(D)J sequence from the TCR mRNA transcript using a triple deoxycytidine stretch at the 3' end of the bead oligo. (Only a single reverse transcribed gene is shown below the drop for simplicity). This deoxycytidine stretch binds to the switch oligo for template switching. Subsequent secondary strand synthesis and PCR amplification complete library preparation.

The present disclosure is compatible with flow cytometry applications including, but not limited to, single cell or bulk cell fluorescence-activated cell sorting (FACS) as well as compatibility with NGS-based applications, multiplex assays and other platforms including single cell assays. The generation of encoded pMHC multimers is described. The present disclosure also describes the preparation of barcoded pMHC via both covalent and non-covalent oligo attachment and the combination of these multimers in the evaluation of pMHC-TCR avidity. The present disclosure also describes a novel pMHC multimer barcoding approach targeting TCR α/β constant genes that simplifies sequencing library preparation by simultaneous processing of TCR sequences and pMHC multimer barcodes. The barcoding oligo length and/or sequence is not fixed and can vary, including the use of various base modifications.

This disclosure describes at least two oligo barcoding strategies for pMHC multimers, and in some embodiments pMHC tetramers. The first uses barcoded poly-A tail oligos that link specific pMHCs with the corresponding T cell transcripts comprising TCR sequences. A second barcoding approach uses oligos bound to streptavidin that target the TCRα and/or TCRβ loci, both oligos bearing tetrameric barcodes. Importantly, when TCRα and TCRβ oligos are used simultaneously, they contain the same tetrameric barcoding information for a given tetramer. Using this approach, all reverse-transcribed TCR sequences contain tetrameric barcode sequences that do not require separate library preparation.

MHC protein

The MHC protein provided and used in the compositions and methods of the present disclosure may be any suitable MHC molecule known in the art for which it is desirable to exchange the peptide originally contained in the MHC protein for another peptide. In general, they have the formula (α-β-P) _n , where n is at least 2, eg between 2-10, eg 4. α is the α chain of a class I or class II MHC protein. β is a β chain, which is defined herein as a β chain for a class II MHC protein or a β ₂ microglobulin for an MHC class I protein. P is a peptide antigen.

MHC proteins can be found in mammalian or avian species, such as primate species, especially humans; rodents including mice, rats and hamsters; rabbit; horses, cattle, dogs and cats; It can be derived from others. For example, the MHC protein may be derived from a human HLA protein or a murine H-2 protein. The HLA protein consists of the class II subunits HLA-DPα, HLA-DPβHLA-DQα, HLA-DQβ, HLA-DRα and HLA-DRβ and the class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. include H-2 proteins include class I subunits H-2K, H-2D, H-2L and class II subunits I-Aα, I-Aβ, I-Eα and I-Eβ and β2-microglobulin. The sequences of some representative MHC proteins are described in Kabat et al. Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp724-815. MHC protein subunits suitable for use in the present invention are soluble forms of normally membrane-bound proteins, prepared as known in the art, for example, by deletion of the transmembrane domain and the cytoplasmic domain.

For class I proteins, the soluble form comprises the α1, α2 and α3 domains. Soluble class II subunits comprise the α1 and α2 domains for the α subunit, and the β1 and β2 domains for the β subunit.

Whether the α and β subunits are produced separately and allowed to combine in vitro to form stable heteroduplex complexes, both subunits can be expressed in a single cell. Methods for producing MHC subunits are known in the art.

To prepare MHC-peptide complexes, subunits can bind antigenic peptides and fold in vitro to form stable heterodimer complexes with intrachain disulfide binding domains. The peptide may be included in the initial folding reaction, or it may be added to the empty heterodimer at a later stage. In the method of the present invention, this will be the exiting peptide. Conditions permissive for folding and binding of subunits to peptides are known in the art. As an example, approximately equimolar amounts of solubilized α and β subunits can be mixed in a urea solution. Refolding is initiated by dilution or dialysis with a urea-free buffer solution. Peptides can be loaded into empty class II heterodimers at about pH 5-5.5 for about 1-3 days followed by neutralization, concentration and buffer exchange. However, the particular folding conditions are not critical to the practice of the present invention.

The monomer complex (α-β-P) (here the monomer) can be multimerized. The resulting multimer is stable over a long period of time. Preferably, the multimer is to be formed by linking the monomer to a multivalent entity via specific attachment sites of the α or β subunit as is known in the art (eg, as described in US Pat. No. 5,635,363). can MHC proteins in monomeric or multimeric form may also be bound to beads or other supports.

Often, the multimeric complex is labeled such that it can be detected directly when used in immunostaining or other methods known in the art, or a secondary labeled immune that will specifically bind the complex as known in the art and described herein. It will be used with reagents. For example, the label is fluorescein isothiocyanate (FITC), rhodamine, Texas Red (Texas Red), phycoerythrin (PE), allophycocyanin (APC) , Brilliant Violet ^TM 421, Brilliant UV ^TM 395, Brilliant Violet ^TM 480, Brilliant Violet ^TM 421 (BV421), Brilliant Blue ^TM 515, APC-R700 or APC-Fire750. In some embodiments, the multimeric complex is labeled with a moiety capable of specifically binding to another moiety. For example, the label can be biotin, streptavidin, an oligonucleotide, or a ligand. Other labels of interest may include dyes, enzymes, chemiluminescent agents, particles, radioactive isotopes, or other directly or indirectly detectable substances.

The compositions disclosed herein may include any suitable MHC protein. Typical MHC proteins and peptides disclosed herein are H-2 Kb monomer, HLA-A ^* 02:01 monomer, HLA-A ^* 24:02 monomer, HLA-A ^* 02:01 tetramers, HLA-A ^* 24:02 tetramers, and H-2 Kb tetramers. However, for example, in accordance with known techniques for predicting the affinity of a peptide for an MHC allele, any MHC allele can be used in the compositions and methods herein in the selection of an appropriate exiting peptide.

Justice:

The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

The term “amino acid” is intended to encompass all molecules, natural or synthetic, that contain both amino acid and acid functionality and may be included in polymers of naturally-occurring amino acids. Typical amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

The term "derived", when used in reference to a rearranged variable region gene "derived" from unrearranged variable region and/or unrearranged variable region gene segments, re-sequences the rearranged variable region gene. refers to the ability to trace up to a series of unrearranged variable region gene segments that have been rearranged to form a gene that aligns and expresses a variable domain (considering splice differences and somatic mutations, if applicable). For example, rearranged variable region genes that have undergone somatic mutation are still derived from unrearranged variable region gene segments. In some embodiments, when an endogenous locus is replaced with a universal light or heavy chain locus, the term "derived" refers to the ability to trace the origin of a sequence to the rearranged locus, even if the sequence has undergone somatic mutation.

"PCR handle" means a constant sequence identical to all primers that permit PCR amplification of a barcode region described herein.

The term “barcoded region” refers to a region comprising a unique nucleotide sequence. The minimum length of this nucleotide sequence depends on the total number of MHC multimers that must be uniquely labeled. For example, a nucleotide sequence of 4 nucleotides in length can have 256 different sequences, which can uniquely label up to 256 MHC multimers. A nucleotide sequence of 6 nucleotides in length can have 4096 different sequences, which can uniquely label up to 4096 MHC multimers. Longer tetrameric sequences can be used to increase throughput.

The term "complementarity" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that adenine residues in a first nucleic acid region are capable of forming specific hydrogen bonds ("base pairs") with residues in a second nucleic acid region that are antiparallel to the first region if the residue is thymine or uracil. . Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if, when the two regions are arranged in an antiparallel manner, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region enemy Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby when the first and second portions are arranged in an antiparallel manner, at least of the nucleotide residues of the first portion About 50%, preferably at least 75%, at least about 90%, or at least 95% are capable of base pairing with the nucleotide residues of the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues of the second portion.

Embodiment 1. The multimeric major histocompatibility complex (MHC) comprises:

one or more MHC molecules linked by backbone molecules; and

At least one nucleic acid molecule linked to a backbone molecule, a central stretch of nucleic acid (barcode region). and a second stretch of nucleic acid having a sequence exhibiting complementarity to the target oligo.

Embodiment 2. The multimeric MHC of embodiment 1, wherein the at least one nucleic acid molecule comprises a 5' PCR handle region, a central barcode region and a 3' poly-A tail region, optionally wherein the at least one nucleic acid molecule comprises a 5' PCR handle region, It contains a central barcode region, a unique molecular identifier (UMI) and a 3' poly-A tail region.

Embodiment 3. The multimeric MHC of embodiment 1 or 2, wherein the poly-A tail comprises at least 10 consecutive adenines.

Embodiment 4. The multimeric MHC of any one of embodiments 1-3, wherein if the matching nucleotide of the target oligo is C, G or A, then the 5' terminal nucleotide of the poly-A tail is G, C or T.

Embodiment 5. The multimeric MHC of any one of embodiments 1-4, wherein at least one nucleic acid molecule is at least 10 nucleotides in length and optionally at least one nucleic acid molecule is 10-200 nucleotides in length.

Embodiment 6. A composition comprising a plurality of subsets of multimeric MHCs according to any one of embodiments 1-5, wherein each subset of multimeric MHCs binds a different peptide and has a corresponding barcode region sequence.

Embodiment 7. A method of linking a particular MHC molecule with a corresponding T cell transcript comprises:

a) linked to a complementary target oligo comprising a plurality of multimeric MHC molecules according to any one of embodiments 1-5, a T cell and a 5' PCR handle, a central cell barcode, a UMI and a 3'-poly-(dT) forming a test sample comprising particles;

b) forming droplets from the test sample such that each droplet contains only one particle and one T cell bound to one or more multimeric MHC molecules;

c) generating a T cell cDNA library and an MHC barcode library from each drop; and

d) sequencing both the T cell mRNA library and the MCH barcode library to link specific MHC molecules with the corresponding T cell transcript.

Embodiment 8. The multimeric MHC comprises:

one or more MHC molecules linked by backbone molecules; and

A nucleic acid molecule comprising at least one nucleic acid molecule linked to said backbone, a central stretch of nucleic acid (barcode region), and a nucleotide sequence complementary to a TCR constant gene.

Embodiment 9. The multimeric MHC of embodiment 8 comprises a nucleic acid molecule of type 1 linked to a backbone; wherein the nucleic acid molecule of type 1 comprises a central barcode region and a nucleotide sequence complementary to a TCRα or TCRβ constant gene.

Embodiment 10. The multimeric MHC of embodiment 8 comprises nucleic acid molecules of type 1 and type 2 linked to a backbone, wherein

A type 1 nucleic acid molecule comprises a central barcode region and a nucleotide sequence complementary to a TCRa constant gene;

The type 2 nucleic acid molecule comprises a nucleotide sequence complementary to a central barcode region and a TCRβ constant gene;

The barcode regions of both types of nucleic acid molecules have the same sequence; Optionally, each type of nucleic acid molecule further comprises a UMI, wherein the UMI sequence of the nucleic acid molecule of the first type is different from the UMI sequence of the nucleic acid molecule of the second type.

Embodiment 11. The multimeric MHC of any one of embodiments 8-10, wherein the nucleic acid molecule comprises a 5' PCR handle, a central barcode region and a 3' nucleotide sequence complementary to a TCR constant gene.

Embodiment 12. The multimeric MHC of any one of embodiments 8-11, wherein the nucleic acid molecule comprises a nucleotide sequence complementary to the 5' end of the TCR constant gene.

Embodiment 13. The multimeric MHC of any one of embodiments 8-12, wherein the TCR constant gene is a TCRα constant gene, a TCRβ constant 1 gene, or a TCRβ constant 2 gene.

Embodiment 14. The multimeric MHC of any one of embodiments 8-13, wherein the 5'-terminal and/or 3'-terminal nucleic acid molecule is linked with a backbone molecule.

Embodiment 15. The multimeric MHC of any one of embodiments 8-14, wherein the nucleic acid molecule further comprises a unique identification molecule (UMI) adjacent the barcode region.

Embodiment 16. The multimeric MHC of any one of embodiments 8-14, wherein the at least one nucleic acid molecule is at least 10 nucleotides in length and optionally the at least one nucleic acid molecule is 10-200 nucleotides in length.

Embodiment 17. A composition comprising a plurality of subsets of multimeric MHCs according to any one of embodiments 8-16, wherein each subset of multimeric MHCs binds a different peptide and has a corresponding barcode region sequence.

Embodiment 18. A method of linking a particular MHC molecule to a corresponding TCRα and/or TCRβ sequence comprises:

a) providing at least one multimeric major biobinding complex according to any one of embodiments 8-16;

b) contacting said multimeric MHC molecule with a T cell;

c) isolating the T cells bound to the multimeric MHC molecule from the cells not bound to the multimeric MHC molecule;

d) lysing the isolated T cells;

e) generating a DNA library in which each DNA molecule comprises a sequence of TCRα and/or TCRβ genes and an MHC barcode; and

f) sequencing the DNA library to link specific MHC molecules to the corresponding TCRα and/or TCRβ sequences.

Embodiment 19. The method of embodiment 18, wherein step c) is accomplished by FACS sorting or magnetic bead-based separation.

Embodiment 20. The method of embodiment 18 or 19, wherein the multimeric MHC molecule is directly or indirectly fluorescently labeled.

Embodiment 21. The method of any one of embodiments 18-20, wherein the T cells bound to the barcoded MHC molecule are mass sorted in a single collection tube.

Embodiment 22. The method of any one of embodiments 18-21, wherein the cognate T cells associated with the barcoded MHC molecule are sorted into individual plate wells as single cells.

Embodiment 23. The multimeric MHC comprises:

two or more MHC molecules linked by a backbone molecule; and

A nucleic acid molecule comprising at least one nucleic acid molecule linked to said backbone, a central stretch of nucleic acid (barcode region), and a template switch oligo sequence.

Embodiment 24. The multimeric MHC of embodiment 23, wherein the nucleic acid molecule comprises a 5' PCR handle, a central barcode region, a UMI and a 3' template switch oligo sequence.

Embodiment 25. The multimeric MHC of embodiment 23 or 24, wherein the template switch oligo sequence comprises a 3' stretch of three riboguanosines.

Embodiment 26. The multimeric MHC of any one of embodiments 23-25, wherein at least one nucleic acid molecule is at least 10 nucleotides in length and optionally at least one nucleic acid molecule is 10-200 nucleotides in length.

Embodiment 27. A composition comprising a plurality of subsets of multimeric MHCs according to any one of embodiments 23-26, wherein each subset of multimeric MHCs binds a different peptide and has a corresponding barcode region sequence.

Embodiment 28. A method of linking a particular MHC molecule to a corresponding TCRα and/or TCRβ sequence comprises:

a) binds to an oligo comprising a plurality of multimeric MHC molecules according to any one of embodiments 23-26, a T cell, and a 3' nucleotide sequence complementary to a 5' PCR handle, a center cell barcode, a UMI and a TCR constant gene forming a test sample including the used beads;

b) forming droplets from the test sample such that each droplet contains only one bead and one T cell bound to one or more multimeric MHC molecules;

c) generating a DNA library, wherein each DNA molecule comprises a sequence of TCRα and/or TCRβ genes, and an MHC barcode; and

d) sequencing the DNA library to link specific MHC molecules to the corresponding TCRα and/or TCRβ sequences.

Embodiment 29. The method of embodiment 28, wherein the beads are selected from hydrogel beads, hard beads and soluble beads.

Embodiment 30. The method of embodiment 28 or 29, wherein the beads are linked to two oligos, wherein the first oligo comprises a 3' nucleotide sequence complementary to a 5' PCR handle, a center cell barcode, UMI and TCRα constant genes. do; the second oligo contains a 3' nucleotide sequence complementary to the 5' PCR handle, central cell barcode, UMI and TCRβ constant genes; The central cell barcodes for the two oligos have identical sequences.

Embodiment 31. The method of any one of embodiments 28-30, wherein step c) of preparing the DNA library comprises reverse transcription of TCR mRNA using MMLV reverse transcriptase.

Embodiment 32. The multimeric MHC or method of any one of the preceding embodiments, wherein the PCR handle enables library preparation of barcode sequences.

Embodiment 33. The multimeric MHC or method of any one of the preceding embodiments, wherein the PCR handle has an i7 adapter sequence.

Embodiment 34. The multimeric MHC or method of any one of the preceding embodiments, wherein the barcode region comprises at least 4 nucleotides.

Embodiment 35. The multimeric MHC or method of any one of the preceding embodiments, wherein the barcode region comprises 6 nucleotides.

Embodiment 36. The multimeric MHC or method of any one of the preceding embodiments, wherein the backbone molecule is selected from the group consisting of polysaccharides, glucans, dextran, streptavidin, and streptameric multimers.

Embodiment 37. The multimeric MHC or method of any one of the preceding embodiments, wherein the MHC molecule is linked to the backbone via a streptavidin-biotin bond, via an MHC heavy chain, or via an MHC light chain (b2M).

Embodiment 38. The multimeric MHC or method of any one of the preceding embodiments, wherein the MHC molecule is linked to the backbone via a streptavidin-biotin bond.

Embodiment 39. The multimeric MHC or method of any one of the preceding embodiments, wherein the multimeric MHC comprises at least four MHC molecules.

Embodiment 40. The multimeric MHC or method of any one of the preceding embodiments, wherein the at least one nucleic acid molecule further comprises a chemical modification.

Embodiment 41. The multimeric MHC or method of any one of the preceding embodiments, wherein the 5' or 3' end of at least one nucleic acid is attached to an amino group via a spacer.

Embodiment 42. The multimeric MHC or method of any one of the preceding embodiments, wherein the spacer is a 6 carbon spacer or a 12 carbon spacer.

Embodiment 43. The multimeric MHC or method of any one of the preceding embodiments, wherein at least one nucleic acid molecule comprises phosphorothioated nucleotides at the 5'-end and/or the 3'-end.

Embodiment 44. The multimeric MHC or method of any one of the preceding embodiments, wherein the linkage between the at least one nucleic acid molecule and the backbone molecule allows for inducible dissociation of the nucleic acid molecule.

Embodiment 45. The multimeric MHC or method of any one of the preceding embodiments, wherein the at least one nucleic acid molecule is linked to the backbone molecule via a disulfide bridge.

Embodiment 46. The multimeric MHC or method of any one of the preceding embodiments, wherein the disulfide bridge comprises a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) modified backbone molecule and 5 '-Amino-modified succinimidyl-4-formylbenzamide analog (S-SS-4FB) is formed by binding to a modified nucleic acid molecule.

Embodiment 47. The multimeric MHC or method of any one of the preceding embodiments, wherein the at least one nucleic acid molecule is linked to the backbone molecule via a photocleavage bond.

Embodiment 48. The multimeric MHC or method of any one of the preceding embodiments, wherein the MHC molecule is an MHC class I and/or MHC class II monomer.

Embodiment 49. The multimeric MHC or method of any one of the preceding embodiments, wherein the MHC molecule is complexed with a peptide.

Embodiment 50. The multimeric MHC or method of any one of the preceding embodiments, wherein the MHC molecule is biotinylated.

Embodiment 51. The multimeric MHC or method of any one of the preceding embodiments, wherein the backbone further comprises one or more labels selected from the group consisting of a fluorescent label, a His-tag, and a metal-ion tag.

Embodiment 52. The multimeric MHC or method of any one of the preceding embodiments, wherein the backbone is directly bound to a fluorescent label.

Embodiment 53. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorescent label is modified with 4FB and is associated with an S-HyNic-modified backbone.

Embodiment 54. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorescent label is a fluorophore-tagged oligo.

Embodiment 55. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorophore-tagged oligo has a 5'-amino or 3'-amino modification and is further modified with 4FB and with an S-HyNic-modified backbone are combined

Embodiment 56. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorophore-tagged oligo is complementary to a nucleic acid molecule linked to the backbone.

Embodiment 57. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorophore-tagged oligo is 10 nucleotides in length.

Embodiment 58. The multimeric MHC or method of any one of the preceding embodiments, wherein the backbone is labeled with a fluorophore labeled anti-streptavidin antibody.

Embodiment 59. The multimeric MHC or method of any one of the preceding embodiments, wherein the fluorophore is a fluorescent dye or quantum dots.

Embodiment 60. The multimeric MHC or method of any one of the preceding embodiments, wherein the at least one nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of DNA, RNA, artificial nucleotides, PNA and LNA.

Embodiment 61. The multimeric MHC or method of any one of the preceding embodiments, wherein the multimeric MHC binds to a cognate T cell.

Embodiment 62. The multimeric MHC or method of any one of the preceding embodiments, wherein the multimeric MHC is compatible with flow cytometry applications.

Embodiment 63. The multimeric MHC or method of any one of the preceding embodiments, wherein the flow cytometry application is single cell or bulk cell fluorescence-activated cell sorting (FACS).

Embodiment 64. The multimeric MHC or method of any one of the preceding embodiments, wherein the multimeric MHC is compatible with NGS-based applications.

Embodiment 65. The multimeric MHC or method of any one of the preceding embodiments, wherein the NGS-based application is droplet-based single cell sequencing.

Embodiment 66. A method of detecting antigen-reactive cells in a sample comprises:

a) providing at least one multimeric major histocompatibility complex according to any one of embodiments 1-5, 8-16, 23-26 and 32-63;

b) contacting said multimeric MHC molecule to said sample; and

c) detecting the binding of the multimeric MHC molecule to the antigen-reactive cell, thereby detecting a cell responsive to the antigen present in the MHC molecule, wherein the binding is linked to one or more MHC molecules via a backbone molecule. is detected by amplifying the barcode region of

Embodiment 67. The method of embodiment 66, wherein the sample is selected from the group consisting of a blood sample, a peripheral blood sample, a blood-derived sample, a tissue sample, bodily fluid, spinal fluid, and saliva.

Embodiment 68. The method of embodiment 66 or 67, wherein the sample is obtained from a mammal.

Embodiment 69. The method of any one of embodiments 66-68, wherein the method is selected from the group consisting of flow cytometry, FACS, magnetic-bead based selection, size-exclusion, gradient centrifugation, column attachment, and gel filtration. further comprising cell selection.

Embodiment 70. The method of any one of embodiments 66-69, wherein the amplification is PCR.

Embodiment 71. The method of any one of embodiments 66-70, wherein detecting the barcode region of the nucleic acid molecule comprises sequencing the barcode region or detecting the barcode region by qPCR.

Example:

The invention, which is now generally described, will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating specific aspects and embodiments of the invention, which are not intended to limit the invention.

Example 1: Construction and Experimental Validation of Barcoded pMHC Multimeric Species

The following describes the covalent and non-covalent association of oligos of any sequence. For covalent bonding, the oligo (eg FIG. 1 ) binds to streptavidin via the formation of a thioether bond, although other chemistries and bond formations may be used. In this particular example, a covalent bond connects both the oligo and streptavidin to a common heterobifunctional linker to create a streptavidin-oligo conjugate ( FIG. 5 ). Oligo non-covalently bound to streptavidin can be achieved by mixing the biotinylated oligo with streptavidin in an optimal ratio and HPLC purification of the desired streptavidin-oligo conjugate species (Fig. 5 and Fig. 9). Covalently and non-covalently derived streptavidin-oligo conjugate species can subsequently be subjected to pMHC multimerization ( FIG. 5 ). These pMHC multimeric species can be used in different combinations for pMHC-TCR avidity studies (as described in FIG. 11 ). Furthermore, a single subtype of pMHC multimer (e.g. only pMHC trimer) has sufficiently high target pMHC-TCR avidity so that differences between 3 and 4 monomers per streptavidin are not known to affect TCR detection. , can be used alone in bulk or single-cell sequencing platforms. The advantage of using non-covalently derived streptavidin-oligo conjugates lies in the cost savings of breaking chemical bonds and using less monomer per streptavidin.

As another example, the Solulink protein-oligonucleotide binding kit can be used with supplemental S-SS-4FB. Streptavidin was first modified with S-HyNic. The 5'-amino modified oligo was modified with S-SS-4FB. The modified oligo and modified streptavidin were then combined to directly barcoded streptavidin ( FIG. 2 ). Alternative binding chemistry approaches and material suppliers can be used to bind barcoded oligos to proteins and allow for inducible barcoded oligo dissociation, including but not limited to photocleavable linkages. Experiments validated the ability of barcoded streptavidin to bind biotin ( FIG. 4 ) as well as the ability to transiently dissociate oligos from streptavidin under reducing conditions ( FIG. 3 ).

Example 2: Fluorophore-based tracking of barcoded tetramers

Barcoded tetramers can be combined with various fluorophore tagging strategies ( FIGS. 6A-6C ) for use in single cell and bulk cell sorting applications.

Example 3: TCR targeting barcoded tetramers

Barcoded pMHC multimers can be prepared to target TCRα and/or TCRβ constant genes ( FIG. 17 ) using the same binding chemistries described. The present disclosure is helpful to scientists interested in obtaining TCR sequences and matching pMHC information. The main advantage of this method is that only one library preparation is required since both reverse-transcribed TCRα and/or TCRβ sequences contain the pMHC multimeric barcode. Thus, a single sequencing read contains both the TCR sequence and pMHC identity information. This is in contrast to poly-A-tail or other capture sequence-based library preparation methods, where the smaller pMHC barcode/antibody barcode library is processed separately and later combined with the mRNA-derived library at sequencing time. The TCR-targeted tetramers described in this disclosure can be combined with fluorophore detection for use in single or bulk cell sorting ( FIGS. 6A-6C ). Single cell sorting allows TCR clonal studies (Figures 18-20) as well as potentially linking TCRα and TCRβ paired sequences to the origin of sorted cells (Figures 18, 21, 22).

Example 4: Custom TCR Droplet-Based Sequencing

In another embodiment, barcoded pMHC multimers can be prepared such that the pMHC multimer-linked oligos comprise a template switch sequence along with a pMHC multimer barcode sequence and a PCR handle sequence ( FIG. 23 ). The key to this is that the switch oligo sequence contains a 3' stretch of 3' riboguanosine to bind deoxycytidines added by MMLV reverse transcriptase. In this scenario, droplet-based beads (including but not limited to hydrogel beads, hard beads or soluble beads) are custom-bound to TCRα and/or TCRβ constant gene complementary oligos using typical cellular barcodes and PCR handle sequences. will be UMI can be included in any oligo sequence for PCR deduplication. Beads with TCRα and TCRβ targeting oligos contain the same barcode for a given bead. Within a single drop containing both beads and pMHC multimer positive T cells, reverse transcription with MMLV reverse transcriptase not only expands bead-based oligos with V(D)J sequences from TCR mRNA, but also pMHC multimer barcodes and Contains a template switching oligo sequence carrying a PCR handle. Subsequent secondary strand synthesis and PCR amplification complete library preparation for sequencing. Oligo sequences, lengths and modifications (including but not limited to the use of locked nucleic acid bases) for bead oligos and pMHC multimeric barcoding/template switching oligos can be altered. This embodiment is compatible with other reverse transcriptase derivatives. This system has several key advantages. One is that only the TCR sequence and the pMHC barcode sequence are obtained, eliminating the need to sequence the entire transcript. Another advantage is that only one library preparation is required since both the tetrameric barcode sequence and the TCR sequence are in the same transcript. Another advantage is that when both TCRα and TCRβ bead oligos are used, both automatically pair because of the unique cellular barcode sequence within each droplet. Finally, only pMHC multimer positive T cells contribute to the sequencing library, as only pMHC multimer positive T cells contain a template switch/pMHC multimer barcoded oligo containing one of the two PCR handles.

Integration by reference

All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, this application, including definitions defined herein, will control.

One. Bentzen, A.K., et al., Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes. Nat Biotechnol, 2016. 34(10): p. 1037-1045.

2. Stoeckius, M., et al., Simultaneous epitope and transcriptome measurement in single cells. Nat Methods, 2017. 14(9): p. 865-868.

3. Peterson, V. M., et al., Multiplexed quantification of proteins and transcripts in single cells. Nat Biotechnol, 2017. 35(10): p. 936-939.

4. Zhang, S. Q., et al., High-throughput determination of the antigen specificities of T cell receptors in single cells. Nat Biotechnol, 2018.

5. Dahotre, S.N., et al., DNA-Barcoded pMHC Tetramers for Detection of Single Antigen-Specific T Cells by Digital PCR. Anal Chem, 2019. 91(4): p. 2695-2700.

6. Altman, J.D. and M.M. Davis, MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells. Curr Protoc Immunol, 2016. 115: p. 17 3 1-17 3 44.

7. Krogsgaard, M. AAI 2019, Abstract 194.58.

8. Krummey, S. M., et al., Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity. J Immunol, 2016. 196(6): p. 2838-46.

9. Zhou, Z.X., et al., Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method. Genome Res, 2013. 23(4): p. 705-15.

equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments encompassed by the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (76)

A peptide-major histocompatibility complex (pMHC) barcoded multimer comprising at least one tunable pMHC entity, wherein the pMHC body comprises a peptide-major histocompatibility complex (pMHC) barcoding Multimer:
at least one pMHC molecule linked by a backbone molecule: and
At least one nucleic acid molecule per backbone molecule comprising a covalently or non-covalently linked binder. The pMHC multimer of claim 1 , wherein the nucleic acid molecule comprises:
a central stretch of pMHC barcoded nucleotides and
A second stretch of nucleotides with complementarity to the target oligo. The pMHC multimer according to claim 2 for use in NGS. 4. The pMHC multimer according to any one of claims 1 to 3, wherein the backbone molecule is streptavidin. 5. The pMHC multimer of any one of claims 1 to 4, wherein the multimer comprises at least 1, at least 2, at least 3 or at least 4 pMHC bodies per backbone molecule. 6. The pMHC multimer according to any one of claims 1 to 5, for use in monitoring T cell receptor (TCR)-pMHC avidity. 7. The pMHC multimer of any one of claims 1 to 6, wherein the streptavidin is covalently bound to at least one nucleic acid molecule to provide at least 4 MHC monomers per streptavidin. 7. The method of any one of claims 1 to 6, wherein the streptavidin is non-covalently bound to at least one nucleic acid molecule, wherein the nucleic acid molecule is biotinylated, at least one biotinylated nucleic acid molecule and strep Tavidin is complexed in a ratio, wherein the ratio is selected from the group consisting of:
1 streptavidin: 1 oligo;
1 streptavidin: 2 oligos, and
1 streptavidin: 3 oligos. The pMHC multimer of claim 1 , wherein the pMHC multimer is prepared by an HPLC purification process. 10. The method of any one of claims 1 to 9, wherein the streptavidin is covalently bound to at least one nucleic acid molecule, the nucleic acid molecule comprising a barcode and at least one biotin binding site, wherein the binding site is biotinylated. A pMHC multimer comprising a peptide, a biotinylated protein, a biotinylated polymer, a biotinylated fluorophore, a biotinylated cleavable oligo or a biotinylated agent. 11. The method of any one of claims 1-10, wherein said at least one nucleic acid molecule consists of a 5' PCR handle region, a central barcode region, optionally a UMI, or optionally a 3' poly-A tail region of at least 10 consecutive adenines. which is a pMHC multimer. 12. The pMHC multimer of any one of claims 1-11, wherein the at least one nucleic acid 3' terminal tail consists of any sequence complementary to a target oligo sequence. 13. The pMHC multimer of any one of claims 1-12, wherein the at least one nucleic acid molecule is about 10-200 nucleotides or more in length. A composition comprising a subset of a plurality of pMHC multimers according to any one of claims 1 to 13, wherein each subset of the pMHC multimers binds a different peptide and has a corresponding barcode region sequence. A method of linking a particular MHC molecule to the corresponding TCRα and/or TCRβ sequence, comprising:
a) forming a test sample comprising a pMHC multimeric molecule according to any one of claims 1 to 13, a T cell, and a particle linked to a binding target oligo comprising a 5' PCR handle, a central cell barcode, a UMI and a bait sequence step;
b) forming droplets from the test sample such that each droplet contains only one particle and one T cell bound to one or more pMHC multimeric molecules;
c) generating a T cell cDNA library and a pMHC barcode library from each drop; and
d) sequencing both the T cell mRNA library and the MCH barcode library to link specific MHC molecules with the corresponding T cell transcript. 16. The method of claim 15, wherein the bait sequence is 3' poly-(dT). Multimeric pMHC comprising:
two or more MHC molecules linked by a backbone molecule; and
A nucleic acid molecule comprising at least one nucleic acid molecule linked to said backbone, a central stretch of nucleic acid (barcode region), and a template switch oligo sequence. 18. The method of claim 17, comprising a type 1 nucleic acid molecule linked to a backbone; wherein the nucleic acid molecule of type 1 comprises a central barcode region and a nucleotide sequence complementary to a TCRα or TCRβ constant gene. 18. The method of claim 17, comprising nucleic acid molecules of type 1 and type 2 linked to a backbone; here
The type 1 nucleic acid molecule comprises a central barcode region and a nucleotide sequence complementary to the TCRα constant gene,
the type 2 nucleic acid molecule comprises a central barcode region and a nucleotide sequence complementary to the TCRβ constant gene;
The barcode regions of the two types of nucleic acid molecules have the same sequence;
optionally wherein the UMI sequences for each of the two types of nucleic acid molecules are random and thus different from each other even though they are located in the same region of each nucleic acid. 20. The multimeric pMHC according to any one of claims 17 to 19, wherein the nucleic acid molecule comprises a 3' nucleotide sequence complementary to a 5' PCR handle, a central barcode region, UMI and TCR constant genes. 21. The multimeric pMHC of any one of claims 17-20, wherein the nucleic acid molecule comprises a nucleotide sequence complementary to the 5' end of the TCR constant gene. 22. The multimeric MHC according to any one of claims 17 to 21, wherein the TCR constant gene is a TCRα constant gene, a TCRβ constant 1 gene or a TCRβ constant 2 gene. 23. The multimeric MHC according to any one of claims 17 to 22, wherein the 5' terminal and/or 3' terminal nucleic acid molecule is linked to a backbone molecule. 24. The multimeric MHC of any one of claims 17-23, wherein the nucleic acid molecule further comprises a unique molecular identifier (UMI) adjacent to the barcode region. 25. The multimeric MHC of any one of claims 17-24, wherein the at least one nucleic acid molecule is about 10-200 nucleotides or more in length. 26. A composition comprising a plurality of multimeric pMHC subsets according to any one of claims 17 to 25, wherein each subset of multimeric MHCs binds a different peptide and has a corresponding barcode region sequence. A method of linking a particular MHC molecule to the corresponding TCRα and/or TCRβ sequence, comprising:
a) providing at least one multimeric pMHC according to any one of claims 17 to 25;
b) contacting said multimeric MHC molecule with a T cell;
c) isolating the T cells bound to the multimeric MHC molecule from the cells not bound to the multimeric MHC molecule;
d) lysing the isolated T cells;
e) generating a DNA library in which each DNA molecule comprises a sequence of TCRα and/or TCRβ genes and a pMHC barcode; and
f) sequencing the DNA library to link specific pMHC molecules to the corresponding TCRα and/or TCRβ sequences. 28. The method of claim 27, wherein step c) is accomplished by FACS sorting or magnetic bead-based separation. 29. The method of claim 27 or 28, wherein the multimeric pMHC molecule is directly or indirectly fluorescently labeled. 30. The method of any one of claims 27-29, wherein the T cells bound to the barcoded pMHC molecule are mass sorted in a single collection tube. 31. The method of any one of claims 27-30, wherein the cognate T cells bound to the barcoded pMHC molecule are sorted into individual plate wells as single cells. Multimeric pMHC comprising:
one or more pMHC molecules linked by backbone molecules; and
A nucleic acid molecule comprising at least one nucleic acid molecule linked to said backbone, a central stretch (barcode region) of a nucleic acid designed to be amplified, and a template switch oligo sequence. 33. The multimeric pMHC of claim 32, wherein the nucleic acid molecule comprises a 5' PCR handle, a central barcode region, a UMI and a 3' template switch oligo sequence. 34. The multimeric pMHC of claim 32 or 33, wherein the template switch oligo sequence comprises a 3' stretch of three riboguanosines. 35. The multimeric pMHC of any one of claims 32-34, wherein the at least one nucleic acid molecule is about 10-200 nucleotides in length or greater. 36. A composition comprising a plurality of multimeric pMHC subsets according to any one of claims 32 to 35, wherein each subset of multimeric pMHCs binds a different peptide and has a corresponding barcode region sequence. A method of linking a particular pMHC molecule to the corresponding TCRα and/or TCRβ sequence, comprising:
a) beads bound to an oligo comprising a plurality of pMHC multimeric molecules according to any one of claims 32 to 35, a T cell and a 3' nucleotide sequence complementary to a 5' PCR handle, a central cell barcode, UMI and TCR constant genes Forming a test sample comprising;
b) forming droplets from the test sample such that each droplet contains only one bead and one T cell bound to one or more multimeric MHC molecules;
c) generating a DNA library in which each DNA molecule comprises a sequence of TCRα and/or TCRβ genes and a pMHC barcode; and
d) sequencing the DNA library to link specific pMHC molecules to the corresponding TCRα and/or TCRβ sequences. 38. The method of claim 37, wherein the beads are selected from hydrogel beads, hard beads and soluble beads. 39. The method of claim 37 or 38, wherein the bead is bound to two oligos, the first oligo comprising a 3' nucleotide sequence complementary to a 5' PCR handle, a central cell barcode, UMI and TCRα constant genes; the second oligo contains a 3' nucleotide sequence complementary to the 5' PCR handle, central cell barcode, UMI and TCRβ constant genes; and the central cell barcodes for the two oligos have the same sequence. 40. The method according to any one of claims 37 to 39, wherein step c) of preparing the DNA library comprises reverse transcription of TCR mRNA using MMLV reverse transcriptase. The multimeric pMHC or method according to any one of the preceding claims, wherein the PCR handle enables the preparation of a library of barcode sequences. The multimeric pMHC or method according to any one of the preceding claims, wherein the PCR handle has an i7 adapter sequence. The multimeric pMHC or method according to any one of the preceding claims, wherein the barcode region comprises at least 4 nucleotides. The multimeric pMHC or method according to any one of the preceding claims, wherein the pMHC molecule is linked to the backbone via a streptavidin-biotin bond, via an MHC heavy chain, or via an MHC light chain (b2M). The multimeric pMHC or method according to any one of the preceding claims, wherein the MHC molecule is linked to the backbone via a streptavidin-biotin bond. The multimeric pMHC or method according to any one of the preceding claims, wherein the multimeric pMHC comprises at least one MHC molecule. The multimeric pMHC or method according to any one of the preceding claims, wherein the at least one nucleic acid molecule further comprises a chemical modification. The multimeric pMHC or method according to any one of the preceding claims, wherein the 5' or 3' end of the at least one nucleic acid is attached to an amino group via a spacer. The multimeric pMHC or method according to any one of the preceding claims, wherein the spacer is a 6-carbon spacer or a 12-carbon spacer. The multimeric pMHC or method according to any one of the preceding claims, wherein the at least one nucleic acid molecule comprises phosphorothioated nucleotides at the 5' and/or 3' ends. The multimeric pMHC or method according to any one of the preceding claims, wherein the linkage between the at least one nucleic acid molecule and the backbone molecule allows for inducible dissociation of the nucleic acid molecule. The multimeric pMHC or method according to any one of the preceding claims, wherein the at least one nucleic acid molecule is linked to the backbone molecule via a covalent or non-covalent bond. The multimeric pMHC or method according to any one of the preceding claims, wherein the covalent bond is a thioether. The multimeric pMHC or method according to any one of the preceding claims, wherein the at least one nucleic acid molecule is linked to the backbone molecule via an inducible cleavable bond. The multimeric pMHC or method according to any one of the preceding claims, wherein the inducibly cleavable bond is photocleavable or comprises a disulfide bond. The multimeric pMHC or method according to any one of the preceding claims, wherein the MHC molecule is an MHC class I and/or an MHC class II monomer. The multimeric pMHC or method according to any one of the preceding claims, wherein the MHC molecule is complexed with a peptide. The multimeric pMHC or method according to any one of the preceding claims, wherein the MHC molecule is biotinylated. The multimeric pMHC or method according to any one of the preceding claims, wherein the backbone further comprises one or more labels selected from the group consisting of a fluorescent label, a His-tag and a metal-ion tag. The multimeric pMHC or method according to any one of the preceding claims, wherein the backbone is directly bound to a fluorescent label. The multimeric pMHC or method according to any one of the preceding claims, wherein the fluorescent label is a fluorophore-tagged oligo. The multimeric pMHC or method according to any one of the preceding claims, wherein the fluorophore-tagged oligo is complementary to a nucleic acid molecule linked to the backbone. The multimeric pMHC or method according to any one of the preceding claims, wherein the backbone is labeled with a fluorophore labeled anti-streptavidin antibody. The multimeric pMHC or method according to any one of the preceding claims, wherein the fluorophore is a fluorescent protein, a fluorescent dye or a quantum dot. The multimeric pMHC or method according to any one of the preceding claims, wherein the at least one nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of DNA, RNA, artificial nucleotides, PNA and LNA. The multimeric pMHC or method according to any one of the preceding claims, wherein the multimeric pMHC binds to a cognate T cell. The multimeric pMHC or method according to any one of the preceding claims, wherein the multimeric pMHC is compatible with flow cytometry applications. The multimeric pMHC or method according to any one of the preceding claims, wherein the flow cytometry application is single cell or bulk cell fluorescence-activated cell sorting (FACS). The multimeric pMHC or method according to any one of the preceding claims, wherein the multimeric pMHC is compatible with NGS-based applications. The multimeric pMHC or method according to any one of the preceding claims, wherein the NGS-based application is droplet-based single cell sequencing. A method for detecting antigen-reactive cells in a sample, comprising:
a) providing at least one multimeric pMHC according to any one of claims 1 to 13;
b) contacting said multimeric pMHC molecule to said sample; and
c) detecting the binding of the multimeric pMHC molecule to the antigen-reactive cell, thereby detecting a cell responsive to the antigen present in the MHC molecule, wherein the binding is linked to one or more MHC molecules via a backbone molecule. is detected by amplifying the barcode region of 72. The method of claim 71, wherein the sample is selected from the group consisting of a blood sample, a peripheral blood sample, a blood-derived sample, a tissue sample, bodily fluid, spinal fluid, and saliva. 73. The method of claims 71 or 72, wherein the sample is obtained from a mammal. 74. The method of any one of claims 1 to 73, wherein the method further comprises selection of cells by a method selected from the group consisting of flow cytometry, FACS, magnetic-bead based selection, size-exclusion, gradient centrifugation, column attachment, and gel filtration. The method comprising 75. The method of any one of claims 1-74, wherein the amplification is PCR. 76. The method of any one of claims 1-75, wherein detecting the barcode region of the nucleic acid molecule comprises sequencing the barcode region or detection of the barcode region by qPCR.

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